This invention relates to microspheres and particularly to fluorescent microspheres and methods of making and using them. Fluorescent microspheres have long been known, but their use has been limited by the complexity of making them, their limited visibility, the limited stability of some forms of them, their tendency to clump together, the lack of sensitivity of procedures using them, and their tendency to produce a large background fluorescence which makes visualization difficult and causes diagnostic error.
Biochemical probes have found widespread use in biochemistry and medicine. A popular review of these technologies is found in Klausner and Wilson, "Gene Detection Technology Opens Doors for Many Industries," Bio/Technology, August 1983, pp. 471-478. The most popular of these probes include a radioactive tracer, or an enzyme which catalyzes color-producing reactions, or a fluorescent species (fluorochrome) which fluoresces brightly under light of the appropriate wavelength. As set out in that article, some of these techniques presently permit detection of as few as 10.sup.4 copies of a single gene, although the techniques require several processing steps and several hours to carry out.
Radioactive probes create substantial handling and waste disposal problems, and color-forming enzymes are insensitive and hence require a large sample of the substrate to visualize.
Fluorochrome labels have now found wide use in microbiology. They are particularly useful in localizing cell surface antigens and in detecting specific nucleic acid sequences, although their usefulness is by no means limited to these applications. Two well-known methods of detecting fluorochrome-labeled species are fluorescence microscopy, described for example in Fluorescent Protein Tracing, ed. by R. C. Nairn, especially Chapter 4 (Churchill Livingstone Ltd. 1976), and flow cytometry, described for example in Muirhead et al., Bio/Technology, 3, pp. 337-356 (1985).
Direct staining with fluorescently-labeled antibodies produces a weak signal which is difficult to detect and presents several other difficulties, such as the complication of the procedure and its tendency to inactivate the antibody during attachment of the fluorescent marker. These difficulties are partially overcome by a sandwich technique in which a fluorescent marker is attached to a secondary antibody that in turn binds to a specific primary antibody. The lack of efficiency and specificity of the secondary binding, however, introduces new problems with this type of indirect method.
A "fluorescent vehicle" for attachment as a visible marker to an IgG fraction of secondary antibody was developed by Molday, Dreyer, Rembaum, and Yen, Journal of Cell Biology, 64, pp. 75-88 (1975). By emulsion copolymerization they synthesized latex microspheres having a diameter from 300 angstroms to 3400 angstroms. These microspheres are designed to be attached to a substrate, namely cell surface antigens to which primary antibodies have been attached, via secondary antibodies bound to the spheres. The spheres may be visualized via electron microscopy or by the addition to the spheres of radioactive or fluorochrome probes. The microspheres contain about 57% methyl ester terminations, about 30% hydroxyethyl ester groups, and about 10% carboxylic acid groups. Fluorescent species were attached to some of these microspheres, having a diameter of 600 angstroms, through the hydroxyethyl ester groups via a cyanogen the carboxylic acid groups. The spheres had limited fluorescence and tended to clump into large aggregates. The spheres were used in an indirect method of localizing cell surface antigens. Variations of these spheres are described in U.S. Pat. Nos. Dreyer, 3,853,987, Yen et al., 4,035,316, Yen et al., 4,105,598, Dreyer, 4,108,972, Rembaum et al., 4,224,198, and Rembaum et al. 4,326,008.
Kaplan et al., Biochimica et Biophysica Acta, 728, pp. 112-120 (1983) modified the Molday et al. technique and synthesized 500 angstrom diameter microspheres containing a fluorescent cross-linking agent, fluorescein. This technique allowed them to incorporate 700 fluorescein molecules per microsphere and reduced background fluorescence. The fluorescent microspheres were then conjugated to acetylated avidin which binds to biotinylated monoclonal antibody using both the sandwich method and direct detection of surface antigens. This involves several steps in addition to the synthesis of fluorescent microspheres, including acetylation of avidin, purification of monoclonal antibody and biotinylation of monoclonal antibody. However, this technique demonstrated the fact that fewer than ten thousand receptors per cell can be detected by fluorescent flow cytometry. These workers suggested the use of larger spheres, which enable them to attach more fluorochrome and thereby enhance the fluorescent signal, to detect cells containing even fewer receptors. Such an approach would also decrease sensitivity because of steric hindrance.
Parks et al., Proc. Natl. Acad. Sci. (USA), 76, pp. 1962-66 (1979), using 0.783 micrometer (7830 angstrom) fluorescent microspheres, showed that as few as some tens of microsphere-antigen conjugates per cell could be used to select hybridomas from mixtures. These authors report that individual beads were visible under laser excitation.
Fuccillo, BioTechniques, 3, pp 494-501 (1985) describes other uses of the avidin-biotin complex.
Fornusek and Vetvicka, "Polymeric Microspheres as Diagnostic Tools for Cell Surface Marker Tracing," in CRC Critical Reviews in Therapeutic Drug Carrier Systems, 2, pp. 137-174 (1986), includes an extensive review of the use of microspheres, including fluorescent microspheres, for cell surface marking. These authors point out that, "Despite all the progress it has brought to cell biology, the fluorescence detection of cell surface markers is relatively tedious and rather insensitive with respect to the recent applications of it." These authors utilize fluorescent microspheres which are on the order of 20,000 angstroms, and cite others who utilize microspheres (Covaspheres.TM.) of about 7,000 to 9,000 angstroms in diameter to make the microspheres individually observable.